Application Of High-pressure Liquid Chromatography Research Articles

Application of HPLC (High Pressure Liquid Chromatography) for Analysis of Lard in the Meatball Product Combined with PCA (Principal Component Analysis)

Meatball is favorite food for Indonesian society. Nowadays, many issues about the food ingredient on meatball were mixed with Lard. Therefore, this study was focused on applications of HPLC UV for the lard detection in meatball. The results showed that the beef and pork meatball can be distinguished using HPLC which was combined Principal Component Analysis (PCA). However, the application of HPLC has a problem for lard detection in meatball because can’t to control the hydrolysis of triglyceride (TGA). Hydrolysis of triglycerides can also be detected using HPLC UV and giving the interference for halal authentication.

Mice Lacking Neutrophil Elastase Are Resistant to Bleomycin-Induced Pulmonary Fibrosis

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.

HPLC−CD On-Line Coupling in Combination with HPLC−NMR and HPLC−MS/MS for the Determination of the Full Absolute Stereostructure of New Metabolites in Plant Extracts

The applicability of on-line coupling of gradient eluted nonchiral reversed-phase high-pressure liquid chromatography (HPLC) to circular dichroism (CD) spectroscopy for the stereochemical analysis of crude plant extracts was investigated. A detector measuring simultaneously UV absorbances and CD effects at constant wavelengths in the same detection cell was used. The solvent system water/acetonitrile was found to be suited to give high-quality CD chromatograms as a function of the elution time and even complete characteristic CD spectra in the stop-flow mode. The method was applied to the tropical liana Habropetalum dawei (Dioncophyllaceae). First, the extract solution was investigated by the combined application of HPLC−NMR and HPLC−ESI-MS/MS techniques. The constitution and relative configuration of two as yet unknown natural products thus detected, the isoquinoline phylline (5) and the naphthylisoquinoline alkaloid 5‘-O-methyldioncopeltine A (6), were characterized. Subsequent HPLC−CD experiments permitted the deduction of the absolute configurations of these new metabolites by empirical analysis of their CD data. Thus, using identical sample solutions and chromatographic conditions (including the same columns and solvent gradient programs), the combination of NMR, MS, and CD data permitted the complete structural elucidation of the two new metabolites in the extract matrix solution, without any isolation and sample purification prior to the coupling experiments.

Application of high-pressure liquid chromatography to studies of collagen production by isolated cells in culture

Techniques for assessing collagen production by cells in culture are usually based on evaluation of uptake of radiolabeled proline into collagen. Although simple in theory, this approach is often flawed because of uncertainties concerning the specific activity of labeled proline in the precursor pool for collagen synthesis. An alternative approach is to assess collagen production directly by measuring hydroxyproline in proteins secreted by cultured cells, although this has been difficult, due to the insensitivity of the methods available. Here we apply high-pressure liquid chromatography using reverse-phase elution of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatives of hydroxyproline to measure collagen production by fibroblasts. The method is easy to perform and allows quantitation of hydroxyproline down to 5 pmol, making it applicable to fibroblasts in 12-well culture plates. Collagen production was shown to be constant over a period of 24 h, with a mean rate of 391 ± 18 (SE n = 14) ng collagen/10 6 cells/h . Similar values were obtained using thin-layer chromatography and an enzyme-linked immunosorbent assay for type I collagen, but these techniques were judged to be less convenient and required additional assumptions compared with the technique described here in full.

Characterization of β-endorphin-immunoreactivity in limbic brain structures of rats self-administering heroin or cocaine

The effects of intravenous self-administration of 30 μg infusions of either heroin or cocaine, or saline on the concentrations of β-endorphin-immunoreactivity (βE-IR) in the anterior part of the rat brain limbic system were studied. Self-administration of heroin and cocaine for 5 daily sessions resulted in a marked reduction of the concentrations of βE-IR in the nucleus accumbens, rostral striatum, septum and hippocampus at the time of the scheduled next session on day 6. In pooled extracts of these regions from rats receiving saline, combined application of high-pressure liquid chromatography (HPLC) fractionation and specific radioimmunoassays revealed the presence of a number of βE-related peptides co-chromatographing with synthetic non-acetylated and acetylated α, β- and γ-type endorphins. Similar profiles were found after HPLC fractionation of extracts of these regions from rats self-administering heroin and cocaine. Rats self-administering heroin or cocaine, however, showed decreased amounts of all detected forms of β-endorphin as compared to saline rats. These findings indicate that both self-administration of an opiate that induces psychic as well as physical dependence and of a non-opiate stimulant inducing psychic but not physical dependence, results in a significant decrease of βE and related peptides in limbic brain regions of the rat. All forms of βE detected after HPLC were equally affected, suggesting an overall effect of the drugs on peptide turnover. These results suggest that βE and related peptides may be involved in the neurochemical mechanisms underlying psychic dependence to drugs.

High‐Pressure Liquid Chromatography (HPLC) of Proteins [New Analytical Methods (29)

AbstractThe application of high‐pressure liquid chromatography (HPLC) to proteins has undergone a dramatic development in recent years. Nowadays its many variants expand the repertoire of high‐performance analysis methods available to the protein chemist, which, until now, have been dominated by electrophoretic techniques. The advent of gene technology has resulted in a renaissance of protein chemistry. The new analytical and preparative problems that have thereby emerged are often ideally solved by HPLC methods. HPLC has long since ceased to be solely a laboratory technique; HPLC systems are now being developed for the separation of proteins–particularly those of great pharmaceutical interest – on a 100‐g scale. The range of applications of analytical and preparative HPLC will be illustrated by two examples of pharmaceutical importance—insulin and interleukin 2.

Ascorbic acid analysis in biological fluids

The application of high-pressure liquid chromatography to quantitative analysis of ascorbic acid in biological samples is described. Experimental conditions were selected that might be representative of those met under a broad range of research and clinical trials. The technique is seen to provide values of ascorbic acid in various samples of guinea pig tissues that are in agreement with an existing analytical method. The short time required for analysis and the use of experimental apparatus that is found with increasing frequency in clinical and research laboratories make this approach attractive. The decay rates of ascorbic acid at different values of temperature and pH are evaluated. Endogenous levels of ascorbic acid in several tissues of fasted and nonfasted guinea pigs are reported.

Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products.

High-pressure liquid chromatography was used to identify the sialo-oligosaccharide products obtained after sialylation of [14C]Gal-GalNAc-protein in vitro by an ovine submaxillary-gland microsomal fraction. Among other products, two isomeric trisaccharides could be identified. NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol and Gal beta 1 leads to 3-(NeuAc alpha 2 leads to 6)GalNAcol respectively, indicating that ovine submaxillary gland contains two sialyltransferases acting on mucin-type acceptors, a beta-galactoside alpha 2 leads to 3 sialyltransferase and a N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. This conclusion was fully supported by methylation analysis of the two trisaccharide products.

Combined application of high-pressure liquid chromatography and field desorption mass spectrometry for the determination of biocides of the phenylurea- and carbamate-type in surface water

A novel method for the indentification and determination of biocides in environmental samples is introduced. The combined application of high-pressure liquid chromatography and field-desorption mass spectrometry has been used for two years to monitor herbicides of the phenylurea-, carbamate- and thiocarbamate-type in Rhine river water. Water samples of 2 liter were used to identify and determine 10 to 150 ng of the biocides (ppt level concentrations). For the HPLC determination a precision of ± 5% and an accuracy of ± 10% was achieved. Although field-desorption mass spectrometry is a costly and somewhat complex analytical technique, the short analyses time (∼10 min), the small sample consumption (∼ng), the high selectivity obtained in multicomponent mixtures and he unmatched reliability for biocide identification are essential advantages.

Application of High-Pressure Liquid Chromatography and Thermal Energy Analyzer to Analysis of Trinitroglycerin and Its Metabolites in Blood

A highly selective and sensitive analytical procedure for the determination of trinitroglycerin and four metabolites in whole blood was developed. Trinitroglycerin and its metabolites were extracted from whole blood with ethyl acetate and analyzed by high-pressure liquid chromatography using the thermal energy analyzer detector. Linearity of response was observed over the 1–1000-ng range. The applicability of this method to the analysis of whole blood from dogs orally dosed with trinitroglycerin is described.

28] High-pressure liquid chromatography of vitamin A metabolites and analogs

Publisher Summary This chapter discusses high-pressure liquid chromatography (HPLC) of vitamin A metabolites and analogs. The application of HPLC to the separation of retinoids has revolutionized vitamin A chromatography and has proved to be an extremely powerful and valuable method for studying vitamin A metabolism. The use of HPLC offers several advantages over conventional methods of vitamin A chromatography, including rapid separation, excellent resolution, quantitative recovery, elimination of artifacts, and the capability of chromatographing a wide range of vitamin A compounds with only minor manipulation of the chromatographic solvent. Straight-phase HPLC on microparticulate silica gel columns has been applied to the study of retinol storage and distribution, retinal isomers and analogs, determination of serum retinol and retinoic acid, and isolation of urinary and fecal metabolites of retinoic acid. This chapter describes the high-pressure liquid chromatography methods developed and used in the authors' laboratory for separation of vitamin A metabolites and analogs. Straight-phase and reverse-phase high-pressure liquid chromatography methods are presented and their application to biochemical problems in the vitamin A field are discussed.

Application of a graphite furnace atomic absorption detector automatically coupled to a high-performance liquid chromatograph for speciation of metal-containing macromolecules

High-pressure liquid chromatography (HPLC) coupled with graphite furnace atomic absorption (GFAA) is capable of sensitive, nearly non-destructive, element-specific separation and detection of a wide range of molecular species containing metals or metalloids. Applications of HPLC—GFAA techniques are discussed, including size exclusion chromatography for the analysis of experimental organometallic polymers containing chemically bonded biocidal organotin moieties, and reversed bonded phase chromatography for analysis of novel organotin silicates. In conjunction with variably sensitive optical refractive index/ultra violet absorption detection, GFAA demonstrates separation of the polymers into at least two tin-containing fractions of widely different molecular weight (MW). The relative proportions of high- and low-MW fractions have important implications with respect to performance specifications for these and similar controlled release materials. Tin-specific and silicon-specific analysis of an organotin silicate demonstrates co-elution of species containing each element. Future off/on-line 29Si and 119Sn Fourier transform nuclear magnetic resonance spectroscopy will demonstrate whether each element is bonded to the same molecular species.

Routine application of HPLC for quantification of aflatoxins in whole peanut kernels

High-pressure liquid chromatography (HPLC) was applied for separation and quantification of the concentration of aflatoxin in 500 samples of shelled unroasted whole peanut kernels. Requirements of the method and reasons for choosing HPLC are discussed, as well as the problems arising from applying the technique to this routine application.

Application of high-pressure liquid chromatography in inorganic analysis

Application of high-pressure liquid chromatography in inorganic analysis

Peptide map analyses of murine Ia antigens of the I–E subregion using HPLC

THE I REGION of the major histocompatibility complex (H–2) of the mouse1 controls various immunologically related functions including immune responsiveness and T-cell, B-cell and macrophage interactions2,3. The only gene products of the I region which have been identified by immunochemical techniques are the Ia antigens which are encoded by loci mapping to the I–A and I–E subregions of the I region4. Previously there has been ambiguity over whether the I–E and/or I–C subregion(s) control molecules detected by immunoprecipitation. Recent evidence suggests that the former, not the latter, controls these molecules5,6. The Ia antigens are highly polymorphic by serological analysis and are expressed predominantly on B lymphocytes and epidermal cells7. Ia molecules are integral cell-surface glycoproteins and consist of two subunits of approximate molecular weights 35,000 (α) and 28,000 (β) which are noncovalently associated7. A general nomenclature has been agreed upon for the Ia polypeptides8. For example, the α polypeptide from the I–E subregion of a mouse of the H–2d haplotype is denoted Eαd. Partial N-terminal amino acid sequence analyses of Ia polypeptides from the I–E and I–A subregions of mice8–14, humans14,15 and guinea pigs16 have led to several important observations. (1) The Eα polypeptides of mice show striking homology with human α chains (p34) at least at their N-termini. The Eβ polypeptides of mice also demonstrate some homology with their guinea pig and human counterparts. (2) The Ia polypeptides from the I–A subregion of mouse show little apparent homology with their human, guinea pig and I–E-subregion counterparts. (3) The Aα, Aβ and Eβ polypeptides of various haplotypes differ by one or more residues at their N-termini. However, for the Eαd and Eαk polypeptides no amino acid differences have been unequivocally demonstrated. To determine whether the N-terminal differences in the Eβ polypeptides extend throughout other regions of these polypeptides and whether the Eα polypeptides are identical, we have undertaken tryptic peptide analyses of two Ia molecules from the I–E subregion. We report here the application of high-pressure liquid chromatography (HPLC) to the separation of tryptic peptides and present comparative tryptic peptide maps for I–E subregion products from the H–2d and H–2k haplotypes.

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.

High-pressure liquid chromatography in polynucleotide synthesis.

Reverse phase high-pressure liquid chromatography (HPLC) using columns containing microparticulate materials with bonded octadecyl groups has been developed as a rapid and efficient method for the separation of nucleosides, nucleotides, and, in particular, of protected oligonucleotides which are standard intermediates in the stepwise synthesis of deoxyribopolynucleotides. Reported are extensive studies of the influence of the different purine and pyrimidine bases, of protecting groups, of the phosphate groups, and of the chain lengths of oligonucleotides on their retention on such columns. Further, the application of HPLC in the stepwise synthesis of an oligonucleotide, d(G-G-A-A-G-C-T-T-A-A-C), has been described. The methods, which are herein described, lend themselves to separations on a preparative scale and effect a marked reduction (up to 50%) in the time required for the synthesis of oligonucleotides.

Application of high-pressure liquid chromatography in inorganic analysis

Die Moglichkeiten zur Reversed-phase-HPLC von Metall-Dithiocarbamaten wurden systematisch untersucht. Auser den Diathyldithiocarbamat-Chelaten wurden in die Untersuchungen die Tetramethylendithiocarbamate einbezogen. Als geeignet und voneinander trennbar erwiesen sich die Diathyldithiocarbamate von Pb, Ni, Co, Cu and Hg sowie die Tetramethylendithiocarbamate von Cd, Pb, Ni, Co, Zn, Cu und Hg. Allen anderen Metallchelate dieser Gruppen zersetzen sich im chromatographischen System, ergeben unsymmetrische oder wegen zu geringer Retention nicht trennbare Banden. Die Ergebnisse aus der Reversed-phase-Chromatographie werden mit den bereits beschriebenen Trennmoglichkeiten mit der Adsorptions-Dunnschicht-Chromatographie und denen der Gas-Chromatographie verglichen und diskutiert.

Forensic aspects of high-pressure liquid chromatography

This paper reviews the applications of high-pressure liquid chromatography (HPLC) to forensic problems, and discusses some of the developments that have taken place in the use of the technique in the Metropolitan Police Laboratory. Preparation of octadecyltrichlorosilane-modified silica is described and some of the chromatographic characteristics of this material are investigated. Applications of HPLC to the analysis of cannabis, opium alkaloids, amphetamine-related materials, LSD and polynuclear hydrocarbons are described.

Biochemical Applications of High-Pressure Liquid Chromatography

Biochemical Applications of High-Pressure Liquid Chromatography